A major goal of bioinorganic chemistry has been to elucidate factors that enable cytochrome P450 to activate inert C-H bonds. Central to these efforts have been attempts to define the geometric and electronic structures of the highly reactive intermediate, termed compound I, that is responsible for these demanding oxidations. Despite considerable effort (measured in both person hours and federal funds) the capture and characterization of P450 compound I (P450-I) proved to be an unattainable goal in biological chemistry for the last forty years. Recently, our laboratory made a breakthrough on this front, discovering how to prepare P450-I in high yield. This discovery opens the door for investigations that could provide key insights into the factors that govern C-H bond activation. Specifically, long-sought electronic and structural characterizations of P450-I are within reach, as are experiments (employing axial-ligand mutants) that directly examine the role of thiolate-ligation in P450 mediated oxidations. It has been hypothesized that P450's electron-donating thiolate ligand promotes C-H bond activation through the generation of basic ferryls. P450-mediated hydroxylations are initiated by H-atom abstraction. Evidence suggests that the ability of metal-oxo complexes to abstract H scales with the strength of the O-H bond formed. In heme proteins, the strength of this O-H bond, D(O-H), is determined by the one-electron reduction potential (E0) of compound I and the pKa of compound II. D(O-H) = 23.06 * E0compound I + 1.37 pKa compound II + 57 2 kcal/mol (1) Eq. 1 highlights the importance of the ferryl pKa and suggests a role for thiolate ligation in P450s: to promote hydrogen abstraction at viable compound I reduction potentials by increasing ferryl basicity. Although experiments have confirmed the basic nature of P450-II, a quantitative measure of this basicity (in the form of a ferryl pKa) has proven elusive. Additionally, E0 has not been determined, complicating estimates of D(O-H). In an exciting turn of events, we have identified a P450 that promises to allow for the determination of a thiolate-ligated ferryl's pKa and reduction potential. Proposed experiments thus provide access to the magnitude of D(O-H), allowing insight into how P450s leverage redox potential and ferryl basicity to activate hydrocarbons. PUBLIC HEALTH RELEVANCE: Cytochrome P450s are responsible for the Phase I metabolism of ~75% of known pharmaceuticals. The capture and characterization of the principal intermediate involved in this process, P450 compound I (P450-I), has served as an unattainable goal in biological chemistry for the last forty years. We have recently determined the means to prepare P450-I and P450-II (its one-electron reduced form) in high purity, a discovery that opens the door for detailed spectroscopic and kinetic characterizations of the (heretofore) elusive ferryl intermediates.